The overall goal of this research program is to explore the principles underlying the coordinated macromolecular interactions that culminate in the assembly of infectious viruses. Since 1981, more than 25 million people have died of AIDS. Although HIV-1, the causative agent of AIDS, has been extensively studied, many stages of its replication cycle remain poorly understood. One essential yet ill-defined stage is retroviral assembly and maturation, and this will be the focus of our research for the next funding cycle. During the late stages of viral replication, newly translated retroviral Gag proteins assemble into spherical immature capsids that are then released from the cell. Concomitant with release, retroviruses undergo a maturation process in which Gag is cleaved by the viral protease into several smaller domains (MA, CA, and NC), triggering dramatic morphological changes that render the particles infectious. Within the mature virion, MA remains associated with the viral envelope, while CA assembles into a mature capsid surrounding a ribonucleoprotein complex formed by the RNA genome and NC. The lack of global symmetry in the immature and mature capsids has thwarted high-resolution structural studies of retroviruses. However, extensive biochemical and genetic studies have led to the development of model systems for Gag and CA that manifest global symmetry. On the basis of low resolution EM structures, models have been proposed for the immature and mature capsids. The overall goal of this project is to use electron cryo-microscopy, image analysis and molecular modeling to derive higher resolution structures of these model systems. We will define the Gag-Gag interactions important for immature capsid formation by characterizing a well-ordered, novel model system for MuLV Gag. We will extend the resolution of two model systems of full-length CA (HIV-1 and MuLV) to beyond 10 A resolution and study small, spherical CA assemblies to examine the structure of putative pentamers CA. These studies will provide a deeper understanding of the molecular interactions in the immature and mature lattices, which will be important for the design of new therapeutic strategies.